1096 6Thorax 1994;49:1096-1098 Effect of acetazolamide and against sodium metabisulphite-induced Thorax: first published as 10.1136/thx.49.11.1096 on 1 November 1994. Downloaded from bronchoconstriction in mild asthma

B J O'Connor, C T Yeo, Y M Chen-Worsdell, P J Barnes, K F Chung

Abstract Because frusemide - but not the more potent Background - Inhaled frusemide but not loop - inhibits indirect bumetanide, another , re- challenges such as MBS and adenosine,5 it duces bronchial responsiveness to sodium seems unlikely that inhibition of the Na+/K+/ metabisulphite (MBS). To investigate 2C1- cotransporter is the underlying mech- whether the effect of frusemide could be anism of action of frusemide. Because fru- mediated through mechanisms other than semide has inhibitory effects on carbonic Na+/K+/Cl- cotransporter inhibition, the anhydrase activity,' we hypothesised that car- effects of amiloride - an inhibitor of bonic anhydrase inhibition with acetazolamide sodium channels in the airway epithelium may inhibit the bronchoconstrictor effect of - and of acetazolamide - a specific in- MBS. We also investigated whether modi- hibitor of carbonic anhydrase - against fication of sodium fluxes across the apical sur- MBS challenge were studied. face ofthe airway epithelium could be involved. Methods - In two separate randomised Amiloride is another diuretic which inhibits double blind placebo controlled studies, the diffusion of sodium ions through sodium 10 subjects with mild asthma attended on channels at the apical surface of airway epi- four separate occasions to inhale 7-5mg thelial cells.7 We therefore studied whether in- amiloride or matched placebo, and 500 mg haled acetazolamide and amiloride could acetazolamide or placebo, immediately attenuate the bronchoconstrictor effects of in- before MBS challenge. The concentration haled MBS in asthmatic subjects. of MBS required to cause a 20% fall in baseline FEVy (PC20) was measured. Results - Amiloride and acetazolamide Methods had no effect on baseline FEV1. Amiloride SUBJECTS had no effect against MBS challenge, but Ten non-smoking subjects with mild asthma http://thorax.bmj.com/ acetazolamide increased -log PC20 from (five men) of mean (SE) age 31'3 (3 7) years, a mean (SE) of 0-75 (0.09) to 0-98 (0.06) 93-1 (4 2)% predicted FEVy (table 1) gave representing a 0 77 (0.24) doubling dose informed consent to participate in this study increase. which was approved by the Royal Brompton Conclusions - These results suggest that Hospital ethics committee. All patients were carbonic anhydrase activity in the airways, taking short-acting inhaled j2 adrenergic agon- but not sodium flux, modulates bronchial ists intermittently for the relief of wheeze, but responsiveness to MBS challenge. The ac- none were on inhaled steroid therapy. None on September 24, 2021 by guest. Protected copyright. tion of frusemide is not likely to involve were smokers. All patients were stable for at inhibition of carbonic anhydrase activity. least six months before entry to study. (Thorax 1994;49:1096-1098) PROTOCOL Two separate randomised double blind placebo Administration of such as frusemide controlled studies were performed. Each sub- and has previously been shown ject participated in both studies. In the first Department of to improve lung function in patients with bron- study subjects inhaled either amiloride (7 5 mg) Thoracic Medicine, chopulmonary dysplasia. l2 More recent studies or matched placebo, followed five minutes later National Heart and have shown that frusemide can attenuate by airway challenge with MBS. The study visits Lung Institute, Royal were at least seven In the Brompton Hospital, bronchoconstrictor responses induced by a separated by days. London SW3 6LY, UK wide range of indirect challenges including ex- second study subjects inhaled either the car- B J O'Connor ercise, allergen, sodium metabisulphite (MBS), bonic anhydrase inhibitor acetazolamide C T Yeo (500 mg) or matched followed five Y M Chen-Worsdell and adenosine in patients with asthma.' Thus, placebo, P J Barnes diuretics may have direct effects on the airways minutes later by MBS challenge. The study K F Chung in addition to improving cardiopulmonary visits were also separated by at least seven days. Reprints will not be function through their diuretic effects. The MBS challenges were performed at the same available. mechanism of action underlying the protective time of the day. Received 18 April 1994 effects of the loop diuretic, frusemide, in the Returned to authors 8 June 1994 airways is unclear. Loop diuretics such as fru- Revised version received semide and bumetanide are potent inhibitors Delivery of acetazolamide and amiloride 13 July 1994 Accepted for publication of the Na+/K+/2Cl- cotransporter system in Acetazolamide solution (Diamox parenteral, 19 July 1994 the ascending loop ofHenle in the renal tubule.4 Lederle Laboratories, Gosport, Hants, UK; Acetazolamide, amilonide and MBS challenge 1097 Table 1 Patient characteristics Table 2 Mean (SE) FEV, (litres) before andfive minutes after inhalation of amiloide or acetazolamide Patient Age Sex Atopy FEVy (n = 10) (Ol predicted) Thorax: first published as 10.1136/thx.49.11.1096 on 1 November 1994. Downloaded from Before After 1 38 F + 81-1 2 51 M + 83-1 Placebo 3-31 (0 36) 3-13 (0 33) 3 26 F + 82-5 Acetazolamide 3-28 (0 33) 3 04 (0-31) 4 21 M + 102-4 5 30 M - 93-1 Placebo 3-28 (0-35) 3 20 (0-34) 6 23 F + 109-3 Amiloride 3-33 (0 34) 3-14 (0-33) 7 22 F + 82-4 8 33 M + 95 5 9 48 F + 87-5 10 21 M + 114-1 after each set of inhalations at each doubling concentration was measured using a dry 1-5 FEV, 7 A *p

100 mg/ml) nebulised at a concentration of Results 50 mg/ml, diluted in 0 15 M NaCl to a total In both studies inhalation of the diluents alone

volume of 10 ml (total dose 500 mg) was de- or of acetazolamide and amiloride caused no http://thorax.bmj.com/ livered. Amiloride hydrochloride was dissolved significant changes in FEV, (table 2). Mean in 5 ml water and 5 ml 0-15 M NaCl to achieve -log PC20 after amiloride (0-78 (0 08)) was a concentration of 1 mg/ml. The solution was not significantly altered compared with that flavoured with xylitol in order to disguise its after placebo (0-83 (0-07)). However, mean taste. A total of 10 ml was also nebulised. -log PC20 increased significantly from 0 75 Appropriate matched diluent placebos were (0 09)) after placebo to 0-98 (0 06)) after ace- used, with 0 15 M NaCl for acetazolamide tazolamide (figure; p<0.02), representing a

placebo and with 0-075 M NaCl and xylitol for 0-77 (0 24) doubling dilution increase. on September 24, 2021 by guest. Protected copyright. amiloride placebo. All solutions were dispensed from an ultra- sonic nebuliser (de Vilbiss 99 Ultraneb, de Discussion Vilbiss, Hendon, UK), which produced part- The aim ofthe study was to determine whether icles with a mass median diameter of4 gim with the mechanism of action of frusemide, which a maximal output of 4 ml/min. Each subject has been shown to inhibit bronchoconstrictor inhaled the solutions for 10 minutes. responses to indirect challenges, may be related to an effect on sodium transport (through a sodium channel inhibitor) or on carbonic anhy- Sodium metabisulphite challenge drase activity. Our results suggest that this is MBS solutions (Sigma Chemical Company, unlikely to be occurring through significant Poole, UK) were freshly made up in 0-9% alterations in Na+ entry channels at the apical saline in doubling concentrations (06 mg/ml to surface of airway epithelial cells because am- 160 mg/ml) 10 minutes before each challenge. iloride was not effective in inhibiting MBS- Aerosols were delivered from a nebuliser at- induced bronchoconstriction. By contrast, tached to a dosimeter (Morgan Nebichech Ne- acetazolamide caused a significant inhibition buliser Controller; PK Morgan Ltd, Kent, suggesting that carbonic anhydrase activity may UK). The nebuliser had an output of 10 gl/puff modulate MBS-induced bronchoconstriction. with a particle size of mass median diameter The dose of amiloride administered was in 3-5 jtm. The dosimeter was set to nebulise for the same range as that given by App and col- one second with a pause time of 10 seconds, leagues who found a significant improvement and the subjects held their breath for five to in mucociliary clearance which occurred within eight seconds after the nebuliser was triggered. the first 10 minutes and was maintained for a Five inhalations at each doubling concentration further 30 minutes after inhalation in patients were taken in succession and three minutes with cystic fibrosis.8 The concentration of am- 1098 O'Connor, Yeo, Chen-Worsdell, Barnes, Chung iloride used would be within that described carbonic anhydrase activity in afferent peri- as possessing inhibitory actions against Na+ pheral nerves ofrodents. 6 Although the precise absorption,9 although the degree of dilution mechanism of MBS-induced broncho- Thorax: first published as 10.1136/thx.49.11.1096 on 1 November 1994. Downloaded from that occurs at the site of action is difficult to constriction is not clear, it is possible that a non- determine. Our data suggest that changes in cholinergic non-adrenergic neural pathway may Na+ transport and in airway hydration are be involved'7'8 and acetazolamide may inhibit not important mechanisms by which frusemide these pathways. inhibits bronchoconstrictor challenges and are similar to those recently reported by Baldwin and colleagues.'0 Our results are also in agree- 1 Kao LC, Warburton D, Cheng MH, Cedeno C, Platzker ACG, Keens TG. Effects of oral diuretics on pulmonary ment with those of Rodwell et al who showed mechanisms in infants with chronic bronchopulmonary that inhaled amiloride did not protect against dysplasia: results of a double-blind cross over sequential trial. Pediatrics 1984;74:37-44. dry air challenge in asthmatic subjects in whom 2 Kao LC, Warburton D, Sargent CW. acutely protection is afforded by frusemide." However, decreases airway resistance in chronic bronchopulmonary dysplasia. Jf Pediatr 1983;103:624-9. the negative results obtained with amiloride 3 Chung KF, O'Connor BJ. Furosemide: a new therapeutic must be interpreted in the light of the fact that indication for asthma? Drugs News and Perspectives 1992; 5:355-60. it was not possible to test higher concentrations 4 Burg M, Stoner L, Cardinal J, Green N. Furosemide: effect of amiloride because of its relative insolubility. on isolated perfused tubules. Am Jf Physiol 1973;225: 119-24. Our studies with acetazolamide demonstrate 5 O'Connor BJ, Chung KF, Chen-Worsdell Y, Fuller RW, that carbonic anhydrase inhibition provides Bames PJ. Effect of inhaled frusemide and bumetanide on adenosine 5' monophosphate and sodium metabisulfite- some protective effect against MBS challenge. induced bronchoconstriction in asthmatic subjects. Am The dose of acetazolamide used afforded ap- Rev Respir Dis 1991;143:1329-33. 6 Stein JH, Wilson CB, Kirkendall WM. Differences in the proximately half the protective effect observed acute effects of furosemide and ethacrynic acid in man. J with 40 mg of inhaled frusemide.'2 We used Lab Clin Med 1968;71:654-5. 7 Benos DJ. Amiloride: a molecular probe ofsodium transport the maximum concentration of acetazolamide in tissues and cells. Am J Physiol 1982;242:C131-45. that could be maintained in solution and ace- 8 App EM, King M, Helfesrieder R, Kohler D, Matthys H. Acute and long-term amiloride inhalation in cystic fibrosis tazolamide is a very potent inhibitor ofcarbonic lung disease. Am Rev Respir Dis 1990;141:605-12. anhydase activity in vitro.'3 Frusemide has pre- 9 Knowles M, Murray G, Shallal F, Askin F, Ranga V, Gatzy M, et al. Bioelectric properties and ion flow across excised viously been shown to possess weak carbonic human bronchi. J Appl Physiol 1984;56:868-77. anhydrase inhibitory activity in the renal tu- 10 Baldwin DR, Grange KL, Pavord I, Knox AJ. The effect of amiloride on the airway response to metabisulphite in bules as judged by its effect on asthma: a negative report. Eur Respir J 1992;5:1189-92. .' However, because frusemide is gen- 11 Rodwell LT, Anderson SD, Dutoit J, Seale JP. Inhaled amiloride does not protect against dry air challenge in erally more effective in inhibiting MBS-induced asthmatic subjects in whom protection is afforded by bronchoconstriction than acetazolamide, it is furosemide. Eur Respir J 1993;6:855-61. 12 Nichol GM, Alton E, Nix A, Geddes DM, Chung KF, unlikely that the inhibitory effect of frusemide Barnes PJ. Effect of inhaled frusemide on metabisulphite can be ascribed to its relatively weaker effect and methacholine-induced bronchoconstriction and nasal

potential difference in asthmatic subjects. Am Rev Respir http://thorax.bmj.com/ as an inhibitor of carbonic anhydrase. The Dis 1990;142:576-80. inhibitory effect of acetazolamide is not only 13 Maren TH. Carbonic anhydrase: chemistry, physiology and inhibition. Physiol Rev 1967;47:595-781. restricted to MBS challenges because a small 14 O'Donnell WI, Rosenberg M, Niven RW, Drazen JM, Israel but significant protective effect against bron- E. Acetazolamide and furosemide attenuate asthma in- duced by hyperventilation of cold, dry air. Am Rev Respir choconstriction induced by cold dry air chal- Dis 1992;146:1518-23. lenge has recently been reported.'4 15 Sun J, Elwood W, Barnes PJ, Chung KF. Effect of diuretics against neurally-mediated contraction of guinea- The mechanism by which inhibition of car- pig airways: contribution of carbonic anhydrase. Am Rev bonic anhydrase activity may be inhibiting Respir Dis 1993;148:902-8. 16 Cammer W, Tansey FA. Immunocytochemical localization MBS-induced bronchoconstriction in asth- of carbonic anhydrase in myelinated fibres in peripheral on September 24, 2021 by guest. Protected copyright. matic patients is not clear. We have recently nerves of rat and mouse. J Histochem Cytochem 1987;35: 865-70. shown that acetazolamide can inhibit non-ad- 17 Nichol GM, Nix A, Chung KF, Barnes PJ. Characterisation renergic non-cholinergic contractile responses ofbronchoconstrictor responses to sodium metabisulphite aerosol in atopic subjects with and without asthma. Thorax induced by electrical stimulation in guinea pig 1989;44:102-7. bronchial strips by preventing the release of 18 Robuschi M, Gamboro G, Spagnotto S, Vaghi A, Bianco S. Inhaled frusemide is highly effective in preventing contractile neuropeptides from sensory nerve ultrasonically nebulised water bronchoconstriction. Pulm endings.'5 Acetazolamide is known to inhibit Pharmacol 1989;1:187-91.